1. Field of the Invention
The present invention generally relates to assays to determine the digestibility of protein-containing compositions. More specifically, the present invention relates to in-vitro digestibility assays using proteases.
2. Description of Related Art
The digestibility of protein-containing human food or non-human animal feed can vary greatly. This is particularly true for food or feed ingredients utilized in food or feed processing. Additionally, variations in digestibility can be magnified during processing of these ingredients.
Processing is often performed during production of protein-containing human food or non-human animal feed to make the food or feed more digestible, more nutritious, more preservable, or more palatable. Common processing steps include heating, storing, cooling, drying, wetting, subjecting to pressure, fermenting, particle-size reducing (e.g. by grinding), constituent separating, preservative adding, and mixing of ingredients.
Processing of protein-containing food or feed can affect the digestibility of the food or feed. For example, improper heat or pressure treatment can adversely affect digestibility of vegetable- or animal-based feed and food ingredients (Dudley-Cash, 1999, .Feedstuffs 71:10; Wang et al., 1998, Poultry Sci. 77:834). Also, digestibility of food or feed can be affected by storage conditions (Culver et al. 1998, J Dairy Sci. 72:2916) and the quality of the new raw ingredients (Wang et al., Id).
Digestibility may be measured using in vivo or in vitro assays. See generally Swaisgood et al., 1981, Adv. Food Nutr. Res. 35:185. Several in vivo digestibility assays have been developed, including the rat true digestibility assay (Chang et al., 1990, J. Agric. Food Chem. 38:1016), ileal nitrogen digestibility assays using pigs (Jaguelin et al., 1994, Proc. 6thInt. Symp. Digestive Physiol. in Pigs, Bad Dobran, Germany, 114) or dogs (Johnson et al., 1998, J. An. Sci. 76:1112), and precision-fed cockerel assays using conventional or cecectomized animals (Han et al., 1990, Poult. Sci. 69:1544; Wang et al., 1998, Poult. Sci. 77:834). Because these in vivo assays are time consuming, expensive, and difficult to perform, several in vitro digestibility assays have been developed.
In vitro digestibility assays utilize proteolytic enzymes to correlate with in vivo protein digestion. Most of these assays utilize mammalian gastric and/or pancreatic and intestinal enzymes to more closely mimic mammalian digestion. Some assays use one enzyme, usually pepsin, to digest the test protein in solution although papain has also been used (Buchanan, 1969, Br. J. Nutr. 23:533; Buchanan and Byers, J. Sci. Food Agric., 20:364). The digestion is followed by an assay for amino acids. More commonly, the in vitro assays utilize more than one enzyme. Results from these multiple enzyme assays usually correlate more closely to in vivo results than single enzyme assays. Examples of these assays are described in Jaguelin et al., 1994, Proc. 6th Int. Symp. Digestive Physiol. in Pigs, Bad Dobran, Germany, 114 and W, McDonough et al., 1990, J. Assoc. Off. Anal. Chem. 73:622.
A variant of these in vitro enzyme digestibility assays, IDEA Immobilized Digestive Enzyme Assay), utilizes enzymes which are covalently immobilized on large-pore diameter (200 nm) glass beads via an amide linkage (Porter et al., 1984, J. Agr. Food Chem. 32:334; Chang et al., 1990, J. Agric. Food Chem. 38:1016). By employing immobilized enzymes, this assay has the following advantages over soluble enzyme assays: autolysis (digestion of the enzymes by the enzymes themselves) is prevented, the stability of the enzymes can be increased, the digest is not contaminated with the enzymes or their autolysis products, the digest is easily separated from the enzyme, and the immobilized enzymes can be used in multiple assays. The IDEA method uses two bioreactors. The first contains immobilized pepsin and the second contains immobilized trypsin, chymotrypsin, and intestinal mucosal peptidases. The bioreactors employ either a recirculating design (Chang et al., 1992, J. Food Biochem. 16:133) or a fluidized bed design (Culver et al., 1989, J. Dairy Sci. 72:2916). Digestion of the test sample proceeds in the pepsin bioreactor at low pH (xcx9c2) for 18-20 hr at 37xc2x0 C. The pH of the pepsin digest is then adjusted to 7.5 with Na2HPO4 then treated with the second biodigester for 24 hr at 37xc2x0 C. The free a amino groups are then determined using o-phthalaldehyde (OPA) in the presence of mercaptoethanol, which yields adducts which absorb strongly at 340 nm (Church et al., 1985, Anal. Biochem. 146:343). This value is compared the total peptide bonds present in the test sample, determined by complete acid hydrolysis of the sample followed by OPA determination. Digestibility is defined as the fraction of the total peptide bonds present which were hydrolyzed by the digesters.
The IDEA assay has proved useful for digestibility determinations of various complex food compositions (Chang et al., 1990, J. Agric. Food Chem., 38:1016; Thresher et al., 1989, Plant Foods for Hum. Nutr. 39:59; Chang et al., 1992, J. Food Biochem. 16:133). However, the assay takes about two days and requires an elaborate heated recirculation system. Thus, while the IDEA assay is simpler and less costly than the in vivo digestibility assays and has several advantages over the in vitro assays employing soluble enzymes, it is still rather difficult to use for routine determination of digestibility of, e.g., raw materials coming into a processing plant, food or feed at various stages of processing, or finished food or feed. The IDEA system would also be prohibitively time consuming and complex for use in conjunction with animal feed processing; it has not been suggested for that use. Therefore, there is a need for digestibility assays suitable for routine use by, e.g., food and feed processors.
Among the several aspects of the present invention may be noted the provision of methods for determining the digestibility of food and non-human-animal feed. A more specific aspect of the invention is the provision of such methods in the form of assays which are more rapid and/or simpler to execute than previously known assays. It is also an aspect of the invention to provide digestibility assays wherein the digestibility of a protein-containing composition is determined by visually comparing the assay results with a standard result. A further aspect of the invention is the provision of methods for determining the acceptability of non-human-animal feed ingredients by rapid and simple digestibility assays.
Briefly, therefore, the present invention is directed to a method of measuring digestibility of a protein-containing composition. The method comprises (a) mixing the protein-containing composition with an aqueous liquid to form an aqueous composition; (b) incubating the aqueous composition with a first immobilized protease in a reaction vessel and causing movement of the aqueous composition relative to the immobilized protease in such a manner that none of the aqueous by liquid is removed from the reaction vessel to metabolize a first portion of the protein-containing composition into it, hydrolyzed peptide bonds; and (c) estimating the hydrolyzed peptide bonds.
Another embodiment is directed to a method of measuring digestibility of a protein-containing composition comprising (a) mixing the protein-containing composition with an aqueous liquid to form an aqueous composition; (b) incubating the aqueous composition with a first immobilized protease in a reaction vessel and causing movement of the aqueous composition relative to the immobilized protease in such a manner that none of the aqueous liquid is removed from the reaction vessel to metabolize a first portion of the protein-containing composition into hydrolyzed peptide bonds;(c) separating the composition from the first immobilized protease; (d) incubating the aqueous composition with at least one additional immobilized protease in a reaction vessel and moving the aqueous composition relative to the additional immobilized protease in a generally nonlinear or multidirectional manner; and (e) estimating the hydrolyzed peptide bonds.
The invention is also directed to a method of measuring digestibility of a protein-containing non-human animal feed ingredient. The method comprises (a) mixing the ingredient with an aqueous, acidic liquid to form an aqueous ingredient; (b) incubating the aqueous ingredient at 37xc2x0 C. with immobilized pepsin in a reaction vessel and causing movement of the aqueous ingredient relative to the immobilized protease in such a manner that none of the aqueous liquid is removed from the reaction vessel to metabolize a first portion of the aqueous ingredient into hydrolyzed peptide bonds; (c) substantially separating the immobilized pepsin from the aqueous ingredient; (d) adjusting the pH of the aqueous ingredient to approximately 7.5; (e) incubating the aqueous ingredient at 37xc2x0 C. with a mixture of immobilized proteases comprising immobilized trypsin, immobilized chymotrypsin, and immobilized intestinal peptidase in a reaction vessel and causing movement of the aqueous ingredient relative to the immobilized proteases as described in (b) to metabolize a second portion of the aqueous ingredient into hydrolyzed peptide bonds; (f) substantially separating the mixture of immobilized proteases from the aqueous ingredient; and (g) estimating the hydrolyzed peptide bonds in the aqueous ingredient.
Additionally, the present invention is directed to a method of measuring digestibility of a protein-containing composition. The method comprises (a) combining the protein-containing composition with an aqueous liquid to form an aqueous composition; (b) incubating the aqueous composition with a first immobilized protease to metabolize a first portion of the protein-containing composition into free amino groups;.and (c) estimating the free xcex1- and/or xcex5-amino group concentration by converting the free amino groups into a colored product wherein the intensity of the colored product is proportional to the quantity of converted free amino groups, and comparing the colored product with at least one, colored standard of a particular intensity representing the intensity of color achieved when a particular quantity of free xcex1- and/or xcex5-amino groups is converted into the colored product.
In another embodiment, the present invention is directed to a kit for measuring digestibility of a protein-containing to composition. The kit comprises (a) a first immobilized An protease; (b) a container for incubating the first immobilized protease with a solubilized protein-containing composition; (c) a reagent for converting free xcex1- and/or xcex5-amino groups into a colored reaction product; and (d) at least one colored standard of a particular intensity representing the intensity of color achieved when treating a particular quantity of free xcex1- and/or xcex5-amino groups with the reagent.
The present invention is also directed to a method for determining whether a process increases the digestibility of a protein-containing composition. The method comprises (a) mixing a first portion of the composition with an aqueous, acidic liquid to form an aqueous composition; (b) incubating the aqueous composition at 37xc2x0 C. with immobilized pepsin in a reaction vessel and causing movement of the aqueous composition relative to the immobilized pepsin in such a manner that none of the aqueous composition is removed from the reaction vessel to metabolize a first portion of the aqueous composition into free amino groups; (c) substantially separating the immobilized pepsin from the aqueous composition; (d) adjusting the pH of the aqueous composition to approximately 7.5; (e) incubating the aqueous composition at 37xc2x0 C. with a mixture of immobilized proteases comprising immobilized trypsin, immobilized chymotrypsin, and immobilized intestinal peptidase in a reaction vessel and causing movement of the aqueous composition relative to the immobilized proteases in such a manner that none of the aqueous composition is removed from the reaction vessel to metabolize a second portion of the aqueous ingredient into free amino groups; (f) substantially separating the mixture of immobilized proteases from the aqueous ingredient; (g) converting the free xcex1- and/or xcex5-amino groups into a colored product, wherein the intensity of the colored product is proportional to the quantity of free amino groups; (h) determining the intensity of the colored product; (i) executing the process on a second portion of the composition to form a processed composition; (j) perform steps (a) through (h) on the processed composition; and (k) comparing the intensity of the colored product resulting from performing steps (a) through (h) on the composition with the intensity of the colored product resulting from performing steps (a) through (h) on the processed composition.
In another embodiment, the present invention is directed to a method for determining the digestibility of a protein-containing composition. The method comprises combining the protein-containing composition with an aqueous liquid to form an aqueous composition and then combining the aqueous composition with a thiol protease to form a reaction composition. The reaction composition is then mixed for a sufficient time to hydrolyze the peptide bonds in the protein-containing composition; and the hydrolyzed peptide bonds in the reaction composition determined. In one embodiment, the thiol protease is papain.
Still another embodiment is directed to a kit for measuring the digestibility. The kit comprises a thiol protease, preferably papain, a container for incubating the thiol protease with a solubilized protein-containing composition, and a reagent for converting free amino groups into a colored reaction product. In addition, the kit may contain at least one color standard of a particular intensity representing the intensity of color achieved when treating a particular quantity of free amino groups with the reagent. Optionally, kits of the present invention can also include instructions for using the kits to determine the digestibility of a protein-containing composition, food or non-human animal feed ingredient.
Additionally, the present invention is directed to a method for determining the acceptability of a non-human animal feed ingredient. The method comprises (a) mixing the ingredient with an aqueous, acidic liquid to form an aqueous ingredient; (b) incubating the aqueous ingredient at 37xc2x0 C. with immobilized pepsin in a reaction vessel and causing movement of the aqueous ingredient relative to the immobilized pepsin in such a manner that none of the aqueous ingredient is removed from the reaction vessel to metabolize a first portion of the aqueous ingredient into free amino groups; (c) substantially separating the immobilized pepsin from the aqueous ingredient; (d) adjusting the pH of the aqueous ingredient to approximately 7.5; (e) incubating the aqueous ingredient at 37xc2x0 C. with a mixture of immobilized proteases comprising immobilized trypsin, immobilized chymotrypsin, and immobilized intestinal peptidase in a reaction vessel and causing movement of the aqueous ingredient relative to the immobilized proteases in such a manner that none of the aqueous ingredient is removed from the reaction vessel to metabolize a second portion of the aqueous ingredient into free amino groups; (f) substantially separating the mixture of immobilized proteases from the aqueous ingredient; (g) converting,the free xcex1- and/or xcex5-amino groups into a colored product, wherein the intensity of the colored product is proportional to the quantity of free amino groups; and (h) comparing the intensity of the colored product to the intensity of a standard, wherein the standard represents the intensity of colored product corresponding to the quantity of free xcex1- and/or xcex5-amino groups equivalent to a digestibility value representing a threshold for acceptability of the animal feed composition.
The present invention is also directed to a method for screening-compositions to determine their effect on digestibility of a protein-containing composition. The method comprises, (a) mixing a first portion of the protein-containing composition with an aqueous liquid to form an aqueous composition; (b) incubating the aqueous composition with a first immobilized protease in a reaction vessel to metabolize a first portion of the protein-containing composition into hydrolyzed peptide bonds; (c) moving the aqueous composition relative to the immobilized protease in a generally non-linear or multidirectional manner; (d) estimating the hydrolyzed peptide bonds in the aqueous composition; (e) mixing at least one additional portion of the protein-containing composition with the composition to be screened; (f) repeating (a) through (d) on the additional portion; and (g) comparing the results obtained between said first portion and said additional portion. A further embodiment of this method comprises before (d) incubating the aqueous composition with a second immobilized protease in a reaction vessel and moving the aqueous composition relative to the immobilized protease in a generally nonlinear or multidirectional manner to further metabolize the protein-containing composition into hydrolyzed peptide bonds.
The invention is further directed to a method for screening compositions to determine their effect on digestibility of a protein-containing composition, comprising (a) combining a first portion of the protein-containing composition with an aqueous liquid to form an aqueous composition; (b) combining the aqueous composition with a thiol protease to form a reaction composition, (c) mixing the reaction composition for a time sufficient to hydrolyze the peptide bonds in the protein-containing composition; (d) estimating the hydrolyzed peptide bonds in the reaction composition; (e) combining at least one additional portion of the protein-containing composition with the composition to be screened; (f) repeating (a) through (d) on the additional portion and (g) comparing the results obtained between the first portion and the additional portion.